Enveloped viruses use specialized protein machinery to fuse their membrane with the membrane of host cells and deliver their genetic material for replication. In influenza virus, the trimeric hemagglutinin (HA) glycoprotein spike is responsible for host cell attachment and membrane fusion. The sequence of events and mechanistic interplay of fusion protein and membranes that lead to fusion have long resisted structural characterization, but through the use of techniques such as cryo-electron tomography (cryo-ET) and structural mass spectrometry that allow native HA and intact virus to be studied under near physiological conditions, we have recently made significant progress in connecting structure, biophysical mechanisms and biological function. Our initial studies focused on the well-characterized X31 H3N2 reassortant virus strain. This work revealed the nature of activation hotspots on the HA fusion protein and elucidated the architecture and sequence of HA-driven membrane remodeling that leads to efficient fusion. The goal of the current proposed project is to apply these powerful approaches to characterize functional variation in HA-mediated membrane fusion by comparing HA from different subtypes and bearing mutations that promote transmission of influenza virus to new hosts. We also will examine the effect of variation in particle morphology on the fusion process, since the M1 matrix protein works together with HA to mediate fusion and it is also the primary determinant of virion morphology. A mechanistic understanding of variations in HA/M1-mediated fusion, linked to a structural framework, will provide a valuable basis for understanding influenza virus pathogenicity, transmission, and evolution. In turn, this information can offer a unique perspective to help guide rational selection of influenza vaccine constructs, immunogen development, and design of HA-targeting inhibitors.